Epidermolysis Bullosa Simplex (EBS) is a debilitating, dominantly inherited skin blistering condition without an effective treatment. We propose to correct the phenotype of EBS-patient keratinocytes and prepare them for autologous transplantation by using a gene targeting approach. This method allows transcription from genes producing toxic proteins to be disrupted by targeted insertion of vector sequences into the mutant allele. Adeno-Associated Virus (AAV) vectors efficiently transduce primary cells in culture, and their single stranded DNA genomes have been shown to recombine with homologous chromosomal sequences. Furthermore, keratinocytes expressing abnormal keratins may be at a growth disadvantage making gene targeting strategies a plausible approach because of the growth advantage conferred to phenotypically corrected cells. The concept of growth differences of cells expressing normal and abnormal keratins is further supported by reports of revertant somatic mosaicism in patients with epidermolysis bullosa suggesting that even infrequent back mutations, or second site suppressor mutations can be therapeutic. Experiments described in this proposal will demonstrate the feasibility of gene targeting as a treatment approach for EBS and serve as a foundation for future studies that may lead to clinical trials for a variety of debilitating skin diseases.